(1997; 248 pages) [French]
3.3 Methods used in determining the purity of reference substances
A consideration of the methods to be employed in examining a chemical reference substance should take account of its method of preparation and its intended use. Such analytical methods may be divided into two broad categories - those that depend solely upon an intrinsic thermodynamic property of the system (e.g. phase solubility analysis and differential scanning calorimetry), and those that require comparison with an external standard (e.g. chromatographic or spectrophotometric methods). Methods in the former group allow the measurement of total impurity levels in absolute terms but provide little information regarding the molecular structure of the contaminants.
3.3.1 Methods based on intrinsic thermodynamic properties
184.108.40.206 Phase solubility analysis. Phase solubility analysis may be employed to detect contaminating substances, including isomeric species, and to estimate their concentration. The coefficient of variation that can be achieved using this method is about 0.2%. It is applicable to most reference substances and uses relatively simple apparatus. In some instances, phase solubility techniques may permit recovery of highly purified crystals of the main component as well as a concentrated solution of the contaminating substances from which they may be isolated for identification by other methods. These fractions provide significant data bearing upon the acceptability of the reference substance. Phase solubility analysis is time-consuming and its execution demands painstaking attention to detail. It is therefore often regarded as unsuitable for routine use in control laboratories, but it has proved of great value in laboratories engaged in the assessment of reference substances. Some factors that may make the method inapplicable are degradation of the substance during the course of analysis, formation of a solid solution, and polymorphism in the main component. In the rare instance where the ratio of the impurity to the main component is the same as the ratio of their respective solubilities in the solvent system employed, the results may lead to erroneous interpretation.
220.127.116.11 Differential scanning calorimetry. Purity estimation by differential scanning calorimetry is based on the determination of the heat of fusion of the sample and the determination of the change in its melting point caused by the impurities present in it. This analytical method can be performed rapidly and is capable of high precision. It is, however, inapplicable if the substance melts with decomposition and this limits its value as a general procedure for purity estimation of reference substances. Like phase solubility analysis, it is also inapplicable where solid solutions are formed.
3.3.2 Methods based on comparison with external standards and other methods
18.104.22.168 Chromatographic methods. Methods of analysis based on chromatographic separation are especially useful for detecting and determining impurities in reference substances. Thin-layer chromatography and gas-liquid chromatography are often used, and high-pressure liquid chromatography is finding increasing application. The individual components separated by chromatographic methods may sometimes be recovered for characterization.
Thin-layer chromatography employs apparatus that is simple and cheap, it is easy to carry out and is readily applicable even in the microgram range. It is frequently capable of separating closely related compounds, such as geometric isomers and the members of a homologous series. All the constituents of the chromatographed reference substance occur somewhere on the chromatogram. However, some constituents may remain on the starting line of the chromatogram, some may move with the solvent front, some may migrate at the same rate as the main component, and some may remain undetected. The usefulness of the method may be greatly enhanced by means of two-dimensional chromatography and by employing a number of different solvent systems and a variety of methods of detection. It is probably the most widely used method for assessing chemical reference substances. The method is, however, rarely applicable on a quantitative basis but has great value in tests devised to limit the concentration of the amounts of impurities. Variations that may be encountered in material used as stationary phases, particularly when they are obtained from different suppliers, may sometimes cause marked differences in the migration of substances in the thin-layer chromatogram.
The resolving power of gas-liquid chromatography and of high-pressure liquid chromatography usually exceeds that of thin-layer chromatography. Both the first two methods also have the advantage of being readily applicable on a quantitative basis, but require more complex equipment. The use of high-pressure liquid chromatography, employing a spectrophotometric method of detection, is of especial value in the examination of reference substances destined to be used in connexion with ultraviolet spectrophotometric assays. Gas-liquid chromatographic methods, on the other hand, are of particular value in detecting and determining volatile impurities, including solvent residues in reference substances.
22.214.171.124 Spectrophotometric methods. Ultraviolet spectrophotometry is a widely used method for determination of purity. Since it depends upon the presence of a characteristic chromophore, it is capable of detecting impurities that contribute excessively to the absorbance value and may indicate the presence of impurities that have negligible absorbance. However, the utility of the method is limited by the small number of absorption maxima in the ultraviolet range, the large numbers of compounds containing similar characteristic chromophores, and the need for reliance on an external reference standard.
As previously noted, infrared spectrophotometry is of less value for detecting impurities. However, it is sometimes useful, for example in determining the proportion of the geometric isomers. Nuclear magnetic resonance spectroscopy is also useful occasionally for the determination of purity.
126.96.36.199 Titrimetric methods. Titrimetric methods provide a valuable means of confirming the identity and purity of a candidate reference substance and have the distinct advantage that they are usually stoichiometric in nature and that the external standard used may be chosen with regard to its suitability as a primary reference material.
188.8.131.52 Optical rotation methods. Many reference substances are optically active and the relative proportion of optical isomers is usually determined by an optical rotation method. The quantitative use of these techniques is well established and can yield results of high precision, depending on the solvent and the wavelength chosen for measurement.
184.108.40.206 Other methods. Other methods, such as gravimetric analysis, electrophoresis, atomic absorption spectroscopy, polarography, and combustion procedures, may be valuable in the determination of purity. Several of the foregoing methods, as well as other techniques, may be used to determine functional groups or elements. The concentration of impurity in the reference substance may then be calculated, using an assumed atomic or molecular weight for the impurity.
3.3.3 General considerations
Whatever methods are used to evaluate chemical reference substances, it is essential that an accurate assessment of the moisture content and the content of other volatile contaminants be made. This may often be achieved in total terms by drying, under defined conditions that are appropriate to the substance being examined. In certain cases, however, this may not be possible, in which case the water content may be determined by Karl Fischer titration and the content of volatile solvents by gas-liquid chromatography. Without an accurate assessment of these values at the time that other determinations are being made, judgements as to the acceptability of the candidate material will be invalid.